The catalysis of NAD(+) reduction by Rhodococcus opacus hydrogenase was investigated by spectroelectrochemistry in a thin layer cell on a platinum electrode. The NADH formation rates were significantly higher than those obtained with the hydrogenase of Alcaligenes eutrophus which has previously been identified as a good catalyst of this electrochemical reduction. As a consequence, for the first time to our knowledge, a well-identified current peak which corresponded to NAD(+) reduction was observed on the voltammograms obtained with a platinum cathode. Thanks to the efficiency of this catalysis, it has been possible to improve the understanding of the mechanism. A two-step mechanism was assumed, according to the structure of the hydrogenase which is composed of two dimers with distinct hydrogenase and diaphorase activity. At high potentials (above - 0.66 V (SCE)) only the diaphorase dimer was reduced by direct electron transfer from the electrode, without intervention of any hydrogen intermediate. For more negative potentials, a reduced hydrogen species adsorbed on the electrode surface was involved in the mechanism. In this case, catalysis followed a more classic catalytic pathway via the hydrogenase dimer and an intramolecular electron transfer to the diaphorase dimer, which reduced NAD(+).